The objective of this project is to uncover the molecular mechanisms of genetic rearrangements. The transposition reaction of bacteriophage Mu is studied as a model system. Critical steps in Mu transposition are a pair of DNA cleavages and strand transfers involving the ends of Mu DNA sequence and a target DNA; these reactions generate a branched DNA intermediate. These chemical reaction steps take place within higher order protein- DNA complexes called transpososomes, the core of which is composed of two Mu-end DNA segments synapsed by a stably bound tetramer of MuA transposase protein. Transpososome assembly is controlled by a number of cofactors: an enhancer type DNA sequence element called IAS that overlaps the Mu operator sequence and the Mu repressor that binds to it, the MuB protein, the E. coli-encoded HU and IHF proteins, ATP, and Mg++. By making use of a simplified transpososome assembly reaction system, we have shown that both the Mu end DNA cleavage and the subsequent strand transfer at one Mu DNA end are catalyzed by the MuA monomers that are bound to the partner Mu DNA end within a transpososome; this explains why Mu DNA end synapsis is a prerequisite for the catalytic steps. Roles of the IAS and MuB ATPase in the transpososome assembly process are currently under investigation by making use of a reaction system in which a transpososome is assembled from short Mu end DNA fragments. MuB ATPase controls each of the early steps of Mu DNA transposition: It assists transpososome assembly, is involved in the target DNA site selection, activates the MuA transposase for strand transfer reaction, and protects transpososome from premature disassembly by ClpX chaperon protein until strand transfer is completed and the transposition intermediate is ready for DNA replication by the host replication proteins. In turn, the functional state of MuB is controlled by the ATPase cycle and by its interaction with MuA. Structural and functional aspects of MuB-DNA complex are currently under investigation by using a variety of physical and biochemical techniques. Efforts are continued toward solving the high-resolution structure of domains of MuA transposase as well as protein-DNA complexes in collaboration with scientists in LCP/NIDDK and at Chicago University.